Efficacy and sequence specific behavior of antisense oligonucleotides (ONs) in biological systems depend upon their resistance to enzymatic degradation. It is therefore essential, when designing potent antisense drugs, to combine features such as high binding affinity and mismatch sensitivity with nuclease resistance. Unmodified phosphodiester antisense oligonucleotides are degraded rapidly in biological fluids containing hydrolytic enzymes (Shaw, J. P.; Kent, K.; Bird, J.; Fishback, J.; Froehler, B. Nucleic Acids Res. 1991, 19, 747-750; Woolf, T. M.; Jennings, C. G. B.; Rebagliati, M; Melton, D. A. Nucleic Acids Res. 1990, 18, 1763-1769), and the first generation of modified antisense oligonucleotide drugs, such as 2′-deoxyphosphorothioate oligonucleotides, were also subject to enzymatic degradation (Maier, M.; Bleicher, K.; Kalthoff, H.; Bayer, E. Biomed. Pept., Proteins Nucleic Acids 1995, 1, 235-241; Agrawal, S.; Temsamani, J.; Tang, J. Y. Proc. Natl. Acad. Sci. 1991, 88, 7595-7599). Extensive stability against the various nucleases present in biological systems can best be achieved by modified oligonucleotides. Since 3′ exonuclease activity is predominantly responsible for enzymatic degradation in serum-containing medium and in various eukaryotic cell lines, modifications located at the 3′-terminus significantly contribute to the nuclease resistance of an oligonucleotide (Shaw, J.-P.; Kent, K.; Bird, J.; Fishback, J.; Froehler, B. Nucleic Acids Res. 1991, 19, 747-750; Maier, M.; Bleicher, K.; Kalthoff, H.; Bayer, E. Biomed. Pept., Proteins Nucleic Acids 1995, 1,235-241).
The ribosyl sugar moiety has also been extensively studied to evaluate the effect its modification has on the properties of oligonucleotides relative to unmodified oligonucleotides. The 2′-position of the sugar moiety is one of the most studied sites for modification. Certain 2′-substituent groups have been shown to increase the lipohpilicity and enhance properties such as binding affinity to target RNA, chemical stability and nuclease resistance of oligonucleotides. Many of the modifications at the 2′-position that show enhanced binding affinity also force the sugar ring into the C3-endo conformation.
One 2′-substituent group that has been shown to enhance the properties of oligonucleotides for antisense applications is the 2′-O—CH2CH2—O—CH3 (2′-O-MOB). This modification in phosphodiester ONs offers about a 2° C. increase in tm/modification relative to 2′-deoxyphosphorothioate ONs. A phosphodiester ON modified with a 2′-O-MOE has about the same nuclease resistance as a 2′-deoxyphosphorothioate ON as shown by the half-life of the full-length oligonucleotide, t1/2.
Although the 2′-position is a commonly used position for antisense applications, modifications of the 3′ and 5′ terminal hydroxyls of an oligonucleotide hav also been shown to be advantageous sites for modifications. Oligonucleotides bearing conjugate groups at these positions have shown improved pharmacokinetic and biodistribution properties including enhanced protein binding
Peptide nucleic acids (PNAs) are compounds that in some respects are analogous to oligonucleotides, but which differ in structure. In peptide nucleic acids, the deoxyribose backbone has been replaced with a backbone having peptide linkages. Each subunit has attached a naturally occurring or non-naturally occurring base. One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. The synthesis of PNAs via preformed monomers was previously described in WO 92/20702 and WO 92/20703, the contents of which are herein incorporated by reference. More recent advances in the structure and synthesis of PNAs are illustrated in WO 93/12129 and U.S. Pat. No. 5,539,082, issued Jul. 23, 1996, the contents of both are incorporated herein by reference. Further, the literature is replete with publications describing synthetic procedures, biological properties and uses of PNAs. For example, PNAs possess the ability to effect strand displacement of double-stranded DNA. Patel, Nature, 1993, 365, 490. Improved synthetic procedures for PNAs have also been described. Nielsen et al., Science, 1991, 254, 1497; and Egholm, J. Am. Chem. Soc., 1992, 114, 1895. PNAs form duplexes and triplexes with complementary DNA or RNA. Knudson et al., Nucleic Acids Research, 1996, 24, 494; Nielsen et al., J. Am. Chem. Soc., 1996, 118, 2287; Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; and Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677.
PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as evidence by their higher melting temperatures(tm). This high thermal stability has been attributed to the neutrality of the PNA backbone, which does not encounter the charge repulsion present in DNA or RNA duplexes. The neutral backbone of the PNA also renders the tm of PNA/DNA(RNA) duplexes practically independent of salt concentration. Thus the PNA/DNA duplex differs from the DNA/DNA duplex interactions which are highly dependent on ionic strength. Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability (see, e.g., Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 9677).
The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti-parallel when the DNA or RNA strand in a 5′ to 3′ orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5′ end of the DNA or RNA and amino end of the PNA is directed towards the 3′ end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are in reverse orientation with respect to the 5′-3′ direction of the DNA or RNA.
Human serum albumin (HSA) is an important protein that plays a crucial role in in vivo distribution and pharmacokinetics of many substances taken up in blood plasma. Human serum albumin is the principal protein found in blood plasma (approximately 60% of the total), that is responsible for much of the plasma colloidal osmotic pressure and serves as a transport protein carrying large organic anions. HSA in blood plasma binds to many endogenous and exogenous compounds with association constants typically in the range of 104 to 106 M−1. As the most abundant protein in plasma (35-50 mg/mL) it plays an important role in the maintenance of blood pH and colloids and transports the drug substances to different parts in the body. It is also an important drug carrier in plasma. The ability of an oligomeric compound to bind to human serum albumin is an important property that determines its ability to distribute to all target tissues of interest.
Diester oligodeoxynucleotides don't show significant binding to HSA, however, phosphorothioate oligodeoxynucleotides bind with micromolar affinity. While phosphorothioate oligonucleotides exhibit both acceptable levels of nuclease resistance and HSA binding suitable for therapeutic applications, their “stickiness” to serum proteins other than HSA results in inhibitory effects of these proteins. Diester linked oligonucleotides modified with selected 2′-substituent groups have improved pharmacological properties and enhanced nuclease resistance. Combining 2′-modifications with the phosphodiester backbone offers a solution to overcome the stickiness of phosphorothioates and also maintain the nuclease resistance. However, these compounds lack the desired level of HSA binding.
Accordingly, it is the object of this invention to provide methods of increasing the binding of modified oligomeric compounds, particular oligonucleotides, oligonucleosides, and PNA's to proteins such as human serum albumin.
It is also the object of this invention to provide compounds that exhibit high binding affinity to target RNA.
Additional objects, advantages and novel features of this invention will become apparent to those skilled in the art upon examination of the following descriptions, figures and claims thereof, which are not intended to be limiting.